Gene duplication and divergence has driven evolutionary innovation in all domains of life. We have learned a great deal from previous bioinformatic, genetic and biochemical investigations that focused on mutations in duplicated genes. However, these approaches miss an important biological reality ? the context in which a newly duplicated gene is evolving. Mutations elsewhere in the genome that improve fitness in the face of an evolutionary challenge may be just as important as mutations in the gene undergoing divergence. Such mutations may rewire metabolic or regulatory networks in ways that boost fitness in the short run, but may sacrifice a previously well-evolved function in the process. We term these ?expedient? mutations. Expedient mutations and mutations in duplicated genes are inextricably intertwined as organisms evolve new genes. We will investigate the role of expedient mutations during evolution of a new protein using a model system in which that novel protein is required for growth. ?argC E. coli cannot synthesize arginine. A point mutation allows E383A ProA (ProA*) to catalyze both its native reaction and the ArgC reaction, albeit poorly. We evolved ?argC proA* E. coli on glucose + proline (conditions in which there is selection only for improved arginine synthesis). Growth rate is improved by amplification of proA*, a mutation that improves the ability of ProA* to catalyze the ArgC reaction, as well as expedient mutations that enhance arginine synthesis by other mechanisms. We will use our ?argC proA* model system to address three aspects of gene duplication and divergence certain to have played a major role in expanding the capabilities of organisms, shaping their genomes, and determining which lineages win and which lose when environmental conditions change. In Aim 1, we will determine which expedient mutations that arose during evolution of the ?argC proA* strain on glucose + proline are detrimental after an efficient replacement for ArgC has evolved, and how they can be repaired. In Aim 2, we will investigate how expedient mutations enhance fitness in the more complex situation when both the original and novel functions of ProA* are required. Finally, in Aim 3, we will address how genome content, gene context and sequence differences between orthologs affect the process of evolution of a replacement for ArgC in four different bacterial species. This work will answer important questions about how new genes have evolved throughout the history of life and in the present due to new selective pressures imposed by anthropogenic pharmaceuticals and pesticides. We will gain a better understanding of the interplay between mutations in a new gene encoding a weak-link enzyme and mutations in the rest of the genome. We will establish what kinds of collateral damage are caused by expedient mutations, and how those expedient mutations are themselves accommodated. Finally, we will gain insight into how differences in microbial genomes affect the potential for evolution of a new enzyme in different bacteria exposed to the same evolutionary challenge.